L-threonine is known to be an essential amino acid, which has been widely used as an additive to animals' fodder and foods and an animal growth stimulator, as well as a component of medical aqueous solutions and other raw material for medicinal products. L-threonine is currently produced by only five companies in advanced countries, including the Ajinomoto Company in Japan, and is two to three times more expensive than lysine that is known to be highly valuable due to it high price of 5,000-6,000 dollars per ton in the international market. Thus, L-threonine has high growth potential in the world market.
L-threonine is currently produced by microbial fermentation techniques, using mainly mutants derived from wild types of microorganisms, including Escherichia coli, the genus Corynebacterium, the genus Brevibacterium, the genus Serratia and the genus Providencia. Examples of these mutants include those having resistance to amino acid analogues or drugs, and their auxotrophs for diamino-pimelic acid, methionine, lysine and isoleucine (Japanese Pat. Publication No. Heisei 2-219582; Korean Pat. Application No. 1998-32951; Appl. Microbiol. Biotechnol., 29:550-553, 1988). However, such mutant strains are disadvantageous in terms of having low L-threonine productivity and being limited to growth on media supplemented with expensive diamino-pimelic acid or isoleucine due to their auxotrophic properties for the diamino-pimelic acid or isoleucine. That is, in the case of using a mutant requiring diamino-pimelic acid for growth, this fermentative production of L-threonine is costly. Likewise, in the case of using an isoleucine auxotroph, a fermentation medium for this auxotroph must be supplemented with expensive isoleucine, resulting in increased production costs of L-threonine.
These problems may be overcome with an isoleucine-leaky mutant. For example, Korean Pat. Publication No. 92-8365 discloses an isoleucine-leaky mutant that does not need isoleucine in its medium and produces higher levels of L-threonine than known strains. However, this classical mutation method is also time-consuming and ineffective in selecting novel bacterial strains capable of producing high levels of L-threonine. In addition, its greatest disadvantage is being limited in improvement of L-threonine productivity.
In this regard, instead of employing auxotrophs, other methods for mass production of L-threonine have been developed. These methods employ metabolic engineering techniques to obtain recombinant L-threonine-producing microorganisms that have increased activity of enzymes participating in the biosynthesis of L-threonine. That is, genes corresponding to enzymes involving in L-threonine metabolism are isolated using genetic recombination techniques, cloned into proper gene vehicles, and introduced into microbial mutants to improve L-threonine productivity of the mutants.
The present inventors previously developed a method of developing a L-threonine producing strain using such metabolic engineering techniques, as disclosed in Korean Pat. Application No. 2001-6976. Briefly, high yields of L-threonine can be achieved by employing a recombinant microorganism comprising (a) one or more chromosomal copies of a ppc gene encoding phosphoenol pyruvate carboxylase (hereinafter, referred to simply as “ppc”), which catalyzes the formation of oxaloacetate (OAA) from phosphoenol pyruvate (PEP) and (b) an operon including genes encoding aspartokinase 1-homoserine dehydrogenase (thrA), homoserine kinase (thrB) and threonine synthase (thrC), which catalyze the biosynthesis of L-threonine from aspartate.
L-threonine is synthesized from aspartate by a multi-step pathway, wherein the aspartate is formed from OAA converted by PPC from PEP. L-threonine biosynthesis is inhibited when glucose is present in relatively high levels in media in comparison with the bacterial growth rate and the overall rate of the tricarboxylic acid (TCA) cycle . In this situation, ppc gene expression is suppressed, while expression of a gene encoding PEP carboxykinase (hereinafter, referred to simply as “pckA”), which catalyzes the conversion of OAA into PEP is increased. The elevated levels of pckA result in the formation of PEP from OAA as the precursor for amino acid biosynthesis, wherein other by-products are synthesized from the PEP (Goldie H. Medina V., Mol. Gen. Genet., 220(2):191-196, 1990; Dan G. Fraenkel., E.coli and Salmonella, 12:142-150, 1996). Therefore, the pckA gene should be essentially inactivated in order to produce L-threonine in high levels by increasing the flux of metabolic pathways responsible for L-threonine synthesis.
On the other hand, several pathways for L-threonine degradation are known, which include the following three pathways. One involves a pathway initiated by threonine dehydrogenase yielding α-amino-β-ketobutyrate. The α-amino-β-ketobutyrate is either converted to acetyl-CoA and glycine or spontaneously degrades to aminoacetone that is converted to pyruvate. The second pathway involves threonine dehydratase yielding α-ketobutyrate which is further catabolized to propionyl-CoA and finally the TCA cycle intermediate, succinyl-CoA. The third pathway utilizes threonine aldolase (Neidhardt F. C. et al. Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM press. Washington D.C., pp369-370). Among them, the threonine dehydratase is an operon that is expressed under hypoxia and high levels of threonine. The present inventors developed a microorganism with improved productivity of L-threonine by specifically inactivating this operon gene (tdcBC) via a genetic recombination technique (Korean Pat. Application No. 2002-015380).
On the other hand, International Pat. Publication No. WO 02/29080 A2 discloses a method of producing L-threonine using a pckA, gene-defective microorganism, which is prepared by introducing it into a wild type strain of the microorganism a recombinant vector carrying a partially deleted pckA gene. However, this microorganism is problematic with respect to production yield of L-threonine because pathways for degradation and intracellular influx of synthesized L-threonine are still activated in the microorganism.